Natural killer cell enhancing factor C

ABSTRACT

A human natural killer cell enhancing factor C and fragments thereof and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptidees by recombinant techniques is disclosed. Further disclosed are antibodies directed against such polypeptides and fragments or portions thereof and methods for producing such antibodies and utilizing such antibodies for therapeutic or diagnostic purposes. Also disclosed are methods for utilizing such polypeptides and/or antibodies for preventing and/or treating viral infections, inflammation, neoplasia and damge from superoxide radicals. Diagnostic assays for identifying mutations in nucleic acid sequence encoding a polypeptide of the present invention and for detecting altered levels of the polypeptide of the present invention for detecting diseases, for example, cancer, are also disclosed.

[0001] This application is a divisional of U.S. application Ser. No.09/407,891, filed Sep. 29, 1999, which is a divisional of U.S.application Ser. No. 08/467,265, filed Jun. 6, 1995, now U.S. Pat. No.6,255,079, issued Jul. 3, 2001, each of which is incorporated byreference in its entirety

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention has been putatively identified as a natural killercell enhancing factor C, sometimes hereinafter referred to as “NKEF C.”The invention also relates to inhibiting the action of suchpolypeptides.

[0003] Natural killer (NK) cells are a subset of lymphocytes capable oflysing a variety of tumor cells without prior activationLymphokine-activated killer (LAK) cells are mainly NK cells activated byinterleukin-2, and are capable of lysing wider ranges of tumor cellswith higher cytotoxic activity. NK cells are proposed to function asnatural surveillance to deter cancer development in the body (Whiteside,T. and Herberman, R. B., Clin. Immunol. Immunopathol., 58:1-23 (1989)and Trinchieri, G., Adv. Immunol., 47:187-376 (1989)). LAK cells, incombination with IL-2, have been used in experimental models and inclinical studies to successfully treat some metastatic tumors(Rosenberg, S. A., et al., N. Engl. J. Med., 316:889-897 (1987)). NKcells are also important controlling viral infection and the regulationof hematopoiesis (Trinchieri (1989), Kiessling, R., et al., Eur. J.Immunol., 7:655-663 (1977), Kiessling, R. and Wigzell, H., Curr. Top.Microbiol. Immunol., 92:107-123 (1981)). Given the important roles ofNK/LAK cells in maintaining the host well-being, it is not surprisingthat their activities are stringently controlled in vivo.

[0004] NK/LAK activity is influenced by various cellular and humoralcomponents in the blood (Golub, S. H., et al., R. E. Schmidt (ed.):Natural Killer Cells: Biology and Clinical Application, pp. 203-207, S.Karger, AG Basel (1990)), for instance, the regulation by red bloodcells (RBC), which enhance NK cytotoxicity against different targetcells (Shau, H., et al., E. Lotzova (ed.): Natural Killer Cells: TheirDefinition, Functions, Lineage and Regulation: pp. 235-349, S. Karger,AG Basel (1993)) and which also upregulate LAK development (Yannelli, J.R., et al., Cancer Res., 48:5696-5700 (1988)).

[0005] Oxdidative stress is an important yet incompletely understoodphenomenon, cells use reactive oxygen species (ROS) to carry outessential functions. Under proper control, ROS initiates conception,cell differentiation and proliferation. If not properly controlled, ROScauses serious damage to cellular components which may lead to apoptoticcell death. ROS are known to cause large-scale cell death, senilechanges, inflammation and tissue injuries in the body.

[0006] Two NKEF genes (NKEF-A and B) from a K562 erythroleukemia cellcDNA library have recently been cloned (Shau, H., et al.,Immunogenetics, 40:129-134 (1994)). They have been identified as membersof a new class of highly conserved antioxidant proteins. They shareextensive homology with each other (88% identical at the amino acidlevel, 71% identical in nucleotide sequence). It is not clear whetherthe dimeric NKEF is a homo- or hetero-dimer of the A or B peptides invivo. NKEF A and NKEF B are differentially expressed in differenttissues. NKEF A and NKEF B have similar antioxidant activity, but NKEF Ahas higher NK enhancing activity than NKEFB. Transfecting NKEF DNA intodifferent cells resulted in cell-type-dependent enhanced cellproliferation or growth inhibition.

[0007] This large family of proposed antioxidant genes are highlyconserved from bacteria to mammals while mammals have been found tocarry more than one NKEF-related gene, bacteria and yeast have beenfound to carry only one copy (Sauri, H., et al.). Members of this familyhave been described as thiol-specific antioxidants. These genes (NKEF-Aand B) encode recombinant proteins which possess antioxidant function inthe protection of protein and DNA from oxidative damage. NKEF is a 44 kDprotein isolated from red blood cell cytosol that increases NK cellcytotoxicity to tumor target cells (Shau, H., et al., Cell. Immunol.,147, 1-11 (1993)). NKEF is a dimer protein composed of two approximately22 kD monomers linked by disulphide bonds.

[0008] Two of the other NKEF-related proteins are human thiol-specificantioxidant protein (HPRP) isolated from a hippocampus cDNA library, andthe proliferation-associated gene (PAG), found to be hyperexpressed intransformed cells. HPRP is 95% identical to NKEF B by nucleotidesequences, and 93% identical by amino acid sequence. Alignment withNKEF-related proteins in other species suggested that NKEF B and HPRPare the same. PAG shares 98% identity with NKEF A by nucleotidesequence, and 97% at the amino acid level, and may be identical toNKEFA.

[0009] In mice, the two homologous genes are MSP23 and MER5. MER5 is 61%identical to NKEF A in amino acid sequence and 64% identical to NKEFB.Even more striking is MSP23, which is 93% identical to NKEF A and 76%identical to NKEFB. MSP23 is induced by oxidative stress in mousemacrophage. MER5 is hyperexpressed in murine erythroleukemic cells, andis necessary for differentiation in those cells. NKEF and NKEF-relatedproteins show no sequence homology to other known antioxidants, such ascatalase, superoxide dismutase, or glutathione peroxidase, nor do theyexhibit the enzymatic activity of those antioxidants.

[0010] This family of antioxidant genes has been found to selectivelysuppress activation of NF- B. Nuclear factor B (NF- B) is atranscriptional activator important for the expression of humanimmunodeficiency virus type I (HIV-I) upon T-cell activating stimuli(Englund, G. et al., Virology, 181:150-157 (1991), Nabel, G., andBaltimore, D., Nature (London), 326:711-713 (1987)). Most of the targetgenes of NF- B in T-cells and other types encode proteins involved inimmune, inflammatory and acute phase responses.

[0011] The polypeptide of the present invention has been putativelyidentified as a natural killer enhancing factor C due to its amino acidsequence homology with human natural killer enhancing factor. Thisidentification has been made as a result of amino acid sequencehomology.

[0012] In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptide of the present invention is ofhuman origin.

[0013] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding a polypeptide ofthe present invention, including mRNAs, DNAs, cDNAs, genomic DNAs aswell as analogs and biologically active and diagnostically ortherapeutically useful fragments thereof.

[0014] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptide byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a nucleic acid sequenceencoding a polypeptide of the present invention, under conditionspromoting expression of said protein and subsequent recovery of saidprotein.

[0015] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptide, orpolynucleotide encoding such polypeptide for therapeutic purposes, forexample, to inhibit the growth of leukemia cells, to treat viralinfection, to augment the effects of natural killer protein to treatneoplasias such as tumors and cancers, to prevent inflammation, and toprevent damage from superoxide radicals in the body, for example, tissueinjury and aging.

[0016] In accordance with yet a further aspect of the present invention,there are also provided nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to a nucleicacid sequence of the present invention.

[0017] In accordance with yet a further aspect of the present invention,there are provided antibodies against such polypeptides.

[0018] In accordance with another aspect of the present invention, thereare provided NKEF C agonist compounds which mimic NKEF C and bind toNKEF C receptors to elicit the biological functions of wild-type NKEF C.

[0019] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedto inhibit the action of such polypeptides, for example, in thetreatment of bone marrow transplant rejection.

[0020] In accordance with still another aspect of the present invention,there are provided diagnostic assays for detecting diseases related tothe expression of the polypeptides and for detecting mutations in thenucleic acid sequences encoding such polypeptides.

[0021] In accordance with yet another aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, as research reagents for invitro purposes related to scientific research, synthesis of DNA andmanufacture of DNA vectors, for the purpose of developing therapeuticsand diagnostics for the treatment of human disease.

[0022] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

[0024]FIG. 1 depicts the cDNA and corresponding deduced amino acidsequence of the polypeptide of the present invention. The standardone-letter abbreviations for amino acids are used. Sequencing wasperformed using a 373 automated DNA sequencer (Applied Biosystems,Inc.).

[0025] FIGS. 2A-D show the amino acid sequence homology between thepolypeptide of the present invention (top comparative line of each row,from SEQ ID NO:2), human NKEF A (second comparative line of each row,SEQ ID NO:14), NKEF B (third comparative line of each row, SEQ IDNO:15), MER5 (fourth comparative line of each row, SEQ ID NO: 16), andMSP23 (fifth comparative line of each row, SEQ ID NO: 17).

[0026]FIG. 3 illustrates the growth inhibitory activity of NKEF Cagainst HL60 human promyelocytic leukemia cells.

[0027]FIG. 4 illustrates the growth inhibitory activity of NKEF Cagainst Jurkat human T-cell leukemia cells.

[0028]FIG. 5 illustrates the effect of NKEF C on VSV lytic infection.

[0029] In accordance with an aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) which encodes for themature polypeptide having the deduced amino acid sequence of FIG. 1 (SEQID NO:2) or for the mature polypeptide encoded by the cDNA of the clonedeposited as ATCC Deposit No. 97157 on May 22, 1995 at the American TypeCulture Collection, ATCC, 10801 University Boulevard, Manassas, Va.20110-2209.

[0030] The polynucleotide of the present invention is highly expressedin heart, liver, skeletal muscle, pancrease, testis, and ovary,moderately expressed in placenta, lung, prostate, small intestine andcolon, and lowly expressed in brain, spleen, thymus and peripheral bloodleukocite. The polynucleotide of this invention was discovered in a cDNAlibrary derived from cyclohexamide treated CEM cells. It is structurallyrelated to a family of highly conserved oxidative stress genes. Itcontains an open reading frame encoding a protein of 271 amino acidresidues of which approximately the first 30 amino acids residues arethe putative leader sequence such that the mature protein comprises 241amino acids. The protein exhibits the highest degree of homology to NKEFB expressed from NK-sensitive erythroleukemic cell line K 562, as shownin Sauri, H., et al. with 68.182% identity and 83.333% similarity overthe entire amino acid stretch. These proteins are significantlyhomologous to several other proteins (thiol-specific antioxidants) froma wide variety of organisms ranging from prokaryotes to mammals,especially with regard to several well-conserved motifs in the aminoacid sequences.

[0031] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIG. 1 (SEQ ID NO:1) or thatof the deposited clone or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same mature polypeptide as the DNA of FIG. 1(SEQ ID NO: 1) or the deposited cDNA.

[0032] The polynucleotide which encodes for the mature polypeptide ofFIG. 1 (SEQ ID NO:2) or for the mature polypeptide encoded by thedeposited cDNA may include, but is not limited to: only the codingsequence for the mature polypeptide; the coding sequence for the maturepolypeptide and additional coding sequence such as a leader or secretorysequence or a proprotein sequence; the coding sequence for the maturepolypeptide (and optionally additional coding sequence) and non-codingsequence, such as introns or non-coding sequence 5′ and/or 3′ of thecoding sequence for the mature polypeptide.

[0033] Thus, the term “polynucleofide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0034] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNAof the deposited clone. The variant of the polynucleotide may be anaturally occurring allelic variant of the polynucleotide or anon-naturally occurring variant of the polynucleotide.

[0035] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the samemature polypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or thepolypeptide encoded by the cDNA of the deposited clone. Such nucleotidevariants include deletion variants, substitution variants and additionor insertion variants.

[0036] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIG. 1 (SEQ ID NO: 1) or of the coding sequence of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

[0037] The present invention also includes polynucleotides, wherein thecoding sequence for the mature polypeptide may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

[0038] Thus, for example, the polynucleotide of the present inventionmay encode for a mature protein, or for a protein having a prosequenceor for a protein having both a prosequence and a presequence (leadersequence).

[0039] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0040] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0041] Fragments of the full length NKEF C gene may be used as ahybridization probe for a cDNA library to isolate the full length geneand to isolate other genes which have a high sequence similarity to theNKEF C gene or similar biological activity. Probes of this typepreferably have at least 30 bases and may contain, for example, 50 ormore bases. The probe may also be used to identify a CDNA clonecorresponding to a full length transcript and a genomic clone or clonesthat contain the complete NKEF C gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the NKEF C gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0042] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO: 1) orthe deposited cDNA(s).

[0043] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, And which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO: 1, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

[0044] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO:2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

[0045] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0046] The present invention further relates to an NKEF C polypeptidewhich has the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) orwhich has the amino acid sequence encoded by the deposited cDNA, as wellas fragments, analogs and derivatives of such polypeptide.

[0047] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIG. 1 (SEQ ID NO:2) or that encoded by the depositedcDNA, means a polypeptide which retains essentially the same biologicalfunction or activity as such polypeptide. Thus, an analog includes aproprotein which can be activated by cleavage of the proprotein portionto produce an active mature polypeptide.

[0048] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0049] The fragment, derivative or analog of the polypeptide of FIG. 1(SEQ ID NO:2) or that encoded by the deposited cDNA may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0050] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0051] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0052] The polypeptides of the present invention include the polypeptideof SEQ ID NO:2 (in particular the mature polypeptide) as well aspolypeptides which have at least 85% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO:2 and more preferably at least90% similarity (more preferably at least 90% identity) to thepolypeptide of SEQ ID NO:2 and still more preferably at least 95%similarity (still more preferably at least 95% identity) to thepolypeptide of SEQ ID NO:2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0053] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0054] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0055] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0056] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes of the present invention. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0057] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0058] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0059] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or P_(L), the phagelambda PL promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0060] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0061] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0062] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0063] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTI, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0064] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0065] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0066] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0067] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0068] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples include the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0069] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), A-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0070] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0071] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMI (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0072] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0073] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0074] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well known to those skilled in the art.

[0075] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0076] The polypeptide can be recovered and purified from recombinantcell cultures by methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0077] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0078] The NKEF C polypeptide of the present invention has been shown tosignificantly augment NK cell-mediated cytotoxicity when added at theinitiation of cytotoxicity assays and NKEF, accordingly, may be employedto regulate NK function.

[0079] The NKEF C polypeptide may be employed to enhance NK activity andtherefore deter cancer development in the body. The NKEF C polypeptidemay also be employed for immunoregulation of NK activity and may beimportant for cells in coping with oxidative insults which leads totissue injury and aging, for example.

[0080] The NKEF C polypeptide of the present invention may also beemployed to prevent inflammation.

[0081] The NKEF C polypeptide of the present invention may also beemployed to prevent NK- B activity and prevent viral transcription andtherefore the proliferation of viral infections. Oxidative stressinduces NF- B activation in T-cells by the transactivator TAX from humanT-cell leukemia type 1 (HDLV-1) and therefore induce viraltranscription. Accordingly, Human immunodeficiency virus type 1 (HIV-1)and HDLV-1 may also be treated with the NKEF C polypeptide of thepresent invention.

[0082] The polypeptide of the present invention may also be employed toinhibit the cytopathic effect of vesicular stromatitis virus and toinhibit the growth of leukemia cells.

[0083] The polynucleotides and polypeptides of the present invention mayalso be employed as research reagents and materials for discovery oftreatments and diagnostics to human disease.

[0084] This invention provides a method for identification of thereceptor for the NKEF C polypeptide. The gene encoding the receptor canbe identified by numerous methods known to those of skill in the art,for example, ligand panning and FACS sorting (Coligan, et al., CurrentProtocols in Immun., 1(2), Chapter 5, (1991)). Preferably, expressioncloning is employed wherein polyadenylated RNA is prepared from a cellresponsive to the NKEF C polypeptide, and a cDNA library created fromthis RNA is divided into pools and used to transfect COS cells or othercells that are not responsive to the NKEF C polypeptide. Transfectedcells which are grown on glass slides are exposed to labeled NKEF Cpolypeptide. The NKEF C polypeptide can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to auto-radiographic analysis. Positive pools areidentified and sub-pools are prepared and re-transfected using aniterative sub-pooling and re-screening process, eventually yielding asingle clone that encodes the putative receptor. As an alternativeapproach for receptor identification, labeled ligand can bephotoaffinity linked with cell membrane or extract preparations thatexpress the receptor molecule. Cross-linked material is resolved by PAGEand exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

[0085] This invention provides a method of screening compounds toidentify those which bind to and activate and those which bind to andinhibit the receptor for the NKEF C polypeptides. As an example, amammalian cell or membrane preparation expressing the NKEF C receptor isincubated with a labeled compound to be tested. The compound may belabeled by a variety of means known in the art, for example, byradioactivity. The ability of the compound to bind to and activate theNKEF C receptor could then be measured by the response of a known secondmessenger system. Such second messenger systems include, but are notlimited to, cAMP guanylate cyclase, ion channels or phosphoinositidehydrolysis. For instance, an effective agonist binds to the NKEF Creceptor and elicits a second messenger response while an effectiveantagonist binds to the receptor but does not elicit a second messengerresponse thereby effectively blocking the receptor.

[0086] Potential antagonists include an antibody, or in some cases, anoligopeptide, which binds to the polypeptide. Alternatively, a potentialantagonist may be a closely related protein which binds to the NKEF Creceptor, however, they are inactive forms of the polypeptide andthereby prevent the action of NKEF C since receptor sites are occupied.

[0087] Another potential antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),thereby preventing transcription and the production of NKEF C. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into NKEF C polypeptide(Antisense-Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of NKEF C.

[0088] Potential antagonists include a small molecule which binds to andoccupies the catalytic site of the polypeptide thereby making thecatalytic site inaccessible to substrate such that normal biologicalactivity is prevented. Examples of small molecules include but are notlimited to small peptides or peptide-like molecules.

[0089] The antagonists may be employed to prevent bone marrow transplantrejection. The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0090] The polypeptides of the present invention and agonist andantagonist compounds may be employed in combination with a suitablepharmaceutical carrier. Such compositions comprise a therapeuticallyeffective amount of the polypeptide or agonist or antagonist compound,and a pharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

[0091] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention or agonist orantagonist compounds may be employed in conjunction with othertherapeutic compounds.

[0092] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral, topical, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 g/kg body weight and in most cases they will be administered inan amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 g/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

[0093] The NKEF C polypeptides and agonists and antagonists which arepolypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

[0094] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral plasmid vector containing RNA encoding a polypeptideof the present invention.

[0095] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

[0096] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0097] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol ml, and -actin promoters). Other viral promoters which maybe employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0098] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the -actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0099] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317,-2,-AM, PA12, T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86,GP+envAml2, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0100] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0101] This invention is also related to the use of the NKEF C gene as adiagnostic. Detection of a mutated form of NKEF C will allow a diagnosisof a disease or a susceptibility to a disease which results fromunderexpression of NKEF C for example, tumors and viral infections.

[0102] Individuals carrying mutations in the human NKEF C gene may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, including, but notlimited to blood, urine, saliva, tissue biopsy and autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986))prior to analysis. RNA or cDNA may also be used for the same purpose. Asan example, PCR primers complementary to the nucleic acid encoding NKEFC can be used to identify and analyze NKEF C mutations. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled NKEF CRNA or alternatively, radiolabeled NKEF C antisense DNA sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

[0103] Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

[0104] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0105] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and SI protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0106] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0107] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0108] The present invention also relates to a diagnostic assay fordetecting altered levels of NKEF C protein in various tissues sinceover-expression compared to normal control tissue samples can detect thepresence of a tumor or viral infection. Assays used to detect levels ofNKEF C protein in a sample derived from a host are well-known to thoseof skill in the art and include radioimmunoassays, competitive-bindingassays, Western Blot analysis and preferably an ELISA assay. An ELISAassay initially comprises preparing an antibody specific to the NKEF Cantigen, preferably a monoclonal antibody. In addition a reporterantibody is prepared against the monoclonal antibody. To the reporterantibody is attached a detectable reagent such as radioactivity,fluorescence or in this example a horseradish peroxidase enzyme. Asample is now removed from a host and incubated on a solid support, e.g.a polystyrene dish, that binds the proteins in the sample. Any freeprotein binding sites on the dish are then covered by incubating with anon-specific protein such as bovine serum albumin. Next, the monoclonalantibody is incubated in the dish during which time the monoclonalantibodies attach to any NKEF C proteins attached to the polystyrenedish. All unbound monoclonal antibody is washed out with buffer. Thereporter antibody linked to horseradish peroxidase is now placed in thedish resulting in binding of the reporter antibody to any monoclonalantibody bound to NKEF. Unattached reporter antibody is then washed out.Peroxidase substrates are then added to the dish and the amount of colordeveloped in a given time period is a measurement of the amount of NKEFC protein present in a given volume of patient sample when comparedagainst a standard curve.

[0109] A competition assay may be employed wherein antibodies specificto NKEF C are attached to a solid support and labeled NKEF C and asample derived from the host are passed over the solid support and theamount of label detected attached to the solid support can be correlatedto a quantity of NKEF C in the sample.

[0110] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0111] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0112] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0113] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAhaving at least 50 or 60 bases. For a review of this technique, seeVerma et al., Human Chromosomes: a Manual of Basic Techniques, PergamonPress, New York (1988).

[0114] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0115] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0116] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0117] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0118] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0119] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0120] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0121] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0122] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0123] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0124] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37 C are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0125] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0126] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0127] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 Ag of approximately equimolar amounts of theDNA fragments to be ligated.

[0128] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1

[0129] Bacterial Expression and Purification of soluble NKEF

[0130] The DNA sequence encoding NKEF, ATCC No. 97157, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′sequences of the NKEF C protein and the vector sequences 3′ to NKEF C.Additional nucleotides corresponding to NKEF C were added to the 5′ and3′ sequences respectively. The 5′ oligonucleotide primers used for thefull length sequence with His-tag has the sequence 5′GCGCGGATCCATGGAGGCGCTGCCCTGCT 3′ (SEQ ID NO:3) contains a BamHIrestriction enzyme site followed by NKEF C coding sequence starting fromthe presumed terminal amino acid of the processed protein and withoutthe His-tag 5′ CGCCCATGGAGGCGCTGCCCCTG 3′ (SEQ ID NO:4) and contains aNcoI site. The 5′ primer used for the NKEF C sequence without the leadersequence and without the His-tag is 5′ CGCCCATGGCTGGAGCTGTGCAGGG 3′ (SEQID NO:7) and has a NcoI site and the 5′ primer for the sequence withoutthe leader sequence and with the His-tag is GCGCGGATCCGCTGGAGCTGTGCAGG3′ (SEQ ID NO:5) and contains a BamHI site. The 3′ primers used were asfollows: 5′ CGCGTCTAGATCAATTCAGTTTATCGAAATACTTCAGC 3′ (SEQ ID NO:6)which contains complementary sequences to an XbaI site followed by NKEFC coding sequence; and 5′ CGCGTCTAGATCAATTCAGTTTATCGAAATACTTCAGC 3′ (SEQID NO:[7] 6. The restriction enzyme sites correspond to the restrictionenzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc.Chatsworth, Calif., 91311). pQE-9 encodes antibiotic resistance(Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatablepromoter operator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-9 was then digested with BamHI and XbaI.The amplified sequences were ligated into pQE-9 and were inserted inframe with the sequence encoding for the histidine tag and the RBS. Theligation mixture was then used to transform E. coli strain M15/rep 4(Qiagen, Inc.) by the procedure described in Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989). M15/rep4 contains multiple copies of the plasmid pREP4, whichexpresses the lacd repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies were selected.Plasmid DNA was isolated and confirmed by restriction analysis. Clonescontaining the desired constructs were grown overnight (O/N) in liquidculture in LB media supplemented with both Amp (100 ug/ml) and Kan (25ug/ml). The O/N culture is used to inoculate a large culture at a ratioof 1:100 to 1:250. The cells were grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalactopyranoside”) was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation. The cell pellet was solubilizedin the chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized NKEF C was purified from this solution by chromatography ona Nickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). NKEF C was eluted from the column in 6 molarguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3molar guanidine HCl, 100 mM sodium phosphate. After incubation in thissolution for 12 hours the protein was dialyzed to 10 mmolar sodiumphosphate.

EXAMPLE 2

[0131] Cloning and Expression of NKEF C Using the Baculovirus ExpressionSystem

[0132] The DNA sequence encoding the full length NKEF C protein, ATCCNo. 97157, was amplified using PCR oligonucleotide primers correspondingto the 5′ and 3′ sequences of the gene:

[0133] For the pA2-gP vector the primers have the sequence 5′CGCGGATCCCGAGGCGCTGCCCCTGC 3′ (SEQ ID NO:8) and contains a BamHirestriction enzyme site (in bold) followed by an efficient signal forthe initiation of translation in eukaryotic cells (Kozak, M., J. Mol.Biol., 196:947-950 (1987) and nucleotides of the NKEF C gene; and the 3′primer has the sequence 5′ CGCGGATCCTCAATTCAGTTTATCGAAATAC 3′ (SEQ IDNO:9) and contains the cleavage site for the restriction endonucleaseBamHi and nucleotides complementary to the 3′ non-translated sequence ofthe NKEF C gene.

[0134] For the pA2 vector the sequences were as follows: 5′CGCGGATCCGCCATCATGGAGGCGCTGCCCCTG 3′ (SEQ ID NO:10) and contains a BamHisite and the 3′ primer is 5′ CGCGGATCCTCAATTCAGTTTATCGAAATCA 3′ (SEQ IDNO: 11) and also contains a BamHI site.

[0135] The amplified sequences were isolated from a 1% agarose gel usinga commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment was then digested with the endonuclease BamHI andpurified again on a 1% agarose gel. This fragment is designated F2.

[0136] The vectors pA2-GP and pA2 (modifications of pVL941 vector,discussed below) are used for the expression of the NKEF C protein usingthe baculovirus expression system (for review see: Summers, M. D. andSmith, G. E. 1987, A manual of methods for baculovirus vectors andinsect cell culture procedures, Texas Agricultural Experimental StationBulletin No. 1555). These expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) followed by the recognition sites for the restrictionendonuclease BamHI. The polyadenylation site of the simian virus (SV)40is used for efficient polyadenylation. For an easy selection ofrecombinant virus the beta-galactosidase gene from E.coli is inserted inthe same orientation as the polyhedrin promoter followed by thepolyadenylation signal of the polyhedrin gene. The polyhedrin sequencesare flanked at both sides by viral sequences for the cell-mediatedhomologous recombination of co-transfected wild-type viral DNA. Manyother baculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIMI (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

[0137] The respective plasmid was digested with the restriction enzymeBamHI and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA was then isolated from a 1% agarosegel using the commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated V2.

[0138] Fragment F2 and the dephosphorylated plasmid V2 were ligated withT4 DNA ligase. E.coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBacNKEF) with the NKEF C geneusing the enzyme BamHI. The sequence of the cloned fragment wasconfirmed by DNA sequencing.

[0139] 5 μg of the plasmid pBacNKEF C was co-transfected with 1.0 μg ofa commercially available linearized baculovirus (“BaculoGold baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0140] 1 μg of BaculoGold virus DNA and 5 μg of the plasmid pBacNKEF Cwere mixed in a sterile well of a microtiter plate containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, MD).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture was added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate was rocked back and forth to mix the newly addedsolution. The plate was then incubated for 5 hours at 27° C. After 5hours the transfection solution was removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum was added.The plate was put back into an incubator and cultivation continued at27° C. for four days.

[0141] After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0142] Four days after the serial dilution, the virus was added to thecells and blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculovirus was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

[0143] Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-NKEF C at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

EXAMPLE 3

[0144] Expression of Recombinant NKEF C in COS cells

[0145] The expression of plasmid, NKEF C HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E.coli replication origin, 4) CMVpromoter followed by a polylinker region, an SV40 intron andpolyadenylation site. A DNA fragment encoding the entire NKEF Cprecursor and a HA tag fused in frame to its 3′ end was cloned into thepolylinker region of the vector, therefore, the recombinant proteinexpression is directed under the CMV promoter. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein aspreviously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M.Connolly, and R. Lerner, 1984, Cell 37:767, (1984)). The infusion of HAtag to the target protein allows easy detection of the recombinantprotein with an antibody that recognizes the HA epitope.

[0146] The plasmid construction strategy is described as follows:

[0147] The DNA sequence encoding NKEF, ATCC No. 97157, was constructedby PCR on the original EST cloned using two primers: the 5′ primer 5′GCGCGGATCCACCATGGAGGCGCTG 3′ (SEQ ID NO:12) contains a BamHI sitefollowed by 12 nucleotides of NKEF C coding sequence starting from theinitiation codon; the 3′ sequence 5′GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAATTCAGTTTATC 3′ (SEQ ID NO:13)contains complementary sequences to an XbaI site, translation stopcodon, HA tag and the last 12 nucleotides of the NKEF C coding sequence(not including the stop codon). Therefore, the PCR product contains aBamHi site, NKEF C coding sequence followed by HA tag fused in frame, atranslation termination stop codon next to the HA tag, and an XbaI site.The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digestedwith BamHi and XbaI restriction enzyme and ligated. The ligation mixturewas transformed into E. coli strain SURE (available from StratageneCloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037)the transformed culture was plated on ampicillin media plates andresistant colonies were selected. Plasmid DNA was isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment. For expression of the recombinant NKEF, COS cellswere transfected with the expression vector by DEAE-DEXTRAN method (J.Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A LaboratoryManual, Cold Spring Laboratory Press, (1989)). The expression of theNKEF C HA protein was detected by radio-labelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells werelabelled for 8 hours with ³⁵S-cysteine two days post transfection.Culture media was then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mMTris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysateand culture media were precipitated with an HA specific monoclonalantibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.

EXAMPLE 4

[0148] Expression via Gene Therapy

[0149] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0150] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0151] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer contains an EcoRI site and the 3′primer contains a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the EcoRI and HimdIII fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is used to transform bacteria HB101,which are then plated onto agar-containing kanamycin for the purpose ofconfirming that the vector had the gene of interest properly inserted.

[0152] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0153] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0154] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

EXAMPLE 5

[0155] Growth Inhibitory Activity of NKEF C Against Human Leukemia Cells

[0156] Two-fold serial dilution of purified NKEF C starting from 100ng/ml were made in RPMI 1640 medium with 0.5% FBS. HL60 or Jurkat cellswere harvested from stationary cultures and washed once with medium.Target cells were suspended at 1×10⁵ cells/ml in medium containing 0.5%FBS, then 0.1 ml aliquots were dispensed into 96-well flat-bottomedmicrotiter plates containing 0.1 ml serially diluted test samples.Incubation was continued for 70 hr. The activity was quantified usingMTS [3(4,5-dimethyl-thiazoyl-2-yl) 5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] Assay. MTSassay is performed by the addition of 20 μl of MTS and phenazinemethosulfate (PMS) solution to 96 well plates (Stock solution wasprepared as described by Promega Technical Bulletin No. 169). During a 3hr. incubation, living cells convert the MTS into the aqueous solubleformazan product. Wells with medium only (no cells) were processed inexactly the same manner as the rest of the wells and were used for blankcontrols. Wells with medium and cells were used as baseline controls.The absorbence at 490 nm was recorded using an ELISA reader and isproportional to the number of viable cells in the wells. Cell growthpromotion (positive percentage) or inhibition (negative percentage), asa percentage compared to baseline control wells (variation between threebaseline control well is less than 5%), calculated for each sampleconcentration, by the formula: OD_(experimental)/OD_(baseline control) X100-100. All determinations were made in triplicate. Mean and SD werecalculated by Microsoft Excel.

EXAMPLE 6

[0157] Antiviral Activity of NKEF C against Vesicular Stomatitis Virus(VSV)

[0158] The cytopathic effect reduction (CPER) assay is employed tomeasure the protective effect of NKEF C on the infection and cytopathicprocess of vesicular stomatitis virus (VSV) to normal human dermalfibroblasts (NHDF) from foreskin (Clonetics). In this experiment weperformed serial dilution of NKEF C at a 1:2 ratio and extended thedilution starting from 3 μg/ml to 6 ng/ml final concentration. Thepositive control employed in this experiment was a recombinant human IFNprotein (expressed in E. Coli), which had a previously determinedspecific activity equal to 4×10⁶ units per 100 μl. In addition, wemaintained a negative (untreated) mock control. Semi-purified (70%)protein isolated from E. Coli expressing the NKEF C protein was employedin this study. The NHDF cells were seeded at 2×10⁴/well and incubatedovernight to reach confluence. These cells were incubated for 12 hoursin the presence of each diluted supernatant and then subsequentlychallenged with VSV at an MOI equal to 1×10⁵ pfu/well. The plates werefurther incubated for 15 hours and then fixed and stained with crystalviolet. The plates were scored for CPE by estimating the percentage ofcells surviving on the -microtiter plate. The figure demonstrates a meaneffective NKEF C concentration equal to 100 ng/ml.

[0159] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 17 918 base pairs nucleic acid single linear cDNA CDS 31..843 1AAGGGAACGT GTTTCTCCCC TCGTTTGGTC ATG GAG GCG CTG CCC CTG CTA GCC 54 MetGlu Ala Leu Pro Leu Leu Ala 1 5 GCG ACA ACT CCG GAC CAC GGC CGC CAC CGAAGG CTG CTT CTG CTG CCG 102 Ala Thr Thr Pro Asp His Gly Arg His Arg ArgLeu Leu Leu Leu Pro 10 15 20 CTA CTG CTG TTC CTG CTG CCG GCT GGA GCT GTGCAG GGC TGG GAG ACA 150 Leu Leu Leu Phe Leu Leu Pro Ala Gly Ala Val GlnGly Trp Glu Thr 25 30 35 40 GAG GAG AGG CCC CGG ACT CGC GAA GAG GAG TGCCAC TTC TAC GCG GGT 198 Glu Glu Arg Pro Arg Thr Arg Glu Glu Glu Cys HisPhe Tyr Ala Gly 45 50 55 GGA CAA GTG TAC CCG GGA GAG GCA TCC CGG GTA TCGGTC GCC GAC CAC 246 Gly Gln Val Tyr Pro Gly Glu Ala Ser Arg Val Ser ValAla Asp His 60 65 70 TCC CTG CAC CTA AGC AAA GCG AAG ATT TCC AAG CCA GCGCCC TAC TGG 294 Ser Leu His Leu Ser Lys Ala Lys Ile Ser Lys Pro Ala ProTyr Trp 75 80 85 GAA GGA ACA GCT GTG ATC GAT GGA GAA TTT AAG GAG CTG AAGTTA ACT 342 Glu Gly Thr Ala Val Ile Asp Gly Glu Phe Lys Glu Leu Lys LeuThr 90 95 100 GAT TAT CGT GGG AAA TAC TTG GTT TTC TTC TTC TAC CCA CTTGAT TTC 390 Asp Tyr Arg Gly Lys Tyr Leu Val Phe Phe Phe Tyr Pro Leu AspPhe 105 110 115 120 ACA TTT GTG TGT CCA ACT GAA ATT ATC GCT TTT GGC GACAGA CTT GAA 438 Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Gly Asp ArgLeu Glu 125 130 135 GAA TTC AGA TCT ATA AAT ACT GAA GTG GTA GCA TGC TCTGTT GAT TCA 486 Glu Phe Arg Ser Ile Asn Thr Glu Val Val Ala Cys Ser ValAsp Ser 140 145 150 CAG TTT ACC CAT TTG GCC TGG ATT AAT ACC CCT CGA AGACAA GGA GGA 534 Gln Phe Thr His Leu Ala Trp Ile Asn Thr Pro Arg Arg GlnGly Gly 155 160 165 CTT GGG CCA ATA AGG ATT CCA CTT CTT TCA GAT TTG ACCCAT CAG ATC 582 Leu Gly Pro Ile Arg Ile Pro Leu Leu Ser Asp Leu Thr HisGln Ile 170 175 180 TCA AAG GAC TAT GGT GTA TAC CTA GAG GAC TCA GGC CACACT CTT AGA 630 Ser Lys Asp Tyr Gly Val Tyr Leu Glu Asp Ser Gly His ThrLeu Arg 185 190 195 200 GGT CTC TTC ATT ATT GAT GAC AAA GGA ATC CTA AGACAA ATT ACT CTG 678 Gly Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu Arg GlnIle Thr Leu 205 210 215 AAT GAT CTT CCT GTG GGT AGA TCA GTG GAT GAG ACACTA CGT TTG GTT 726 Asn Asp Leu Pro Val Gly Arg Ser Val Asp Glu Thr LeuArg Leu Val 220 225 230 CAA GCA TTC CAG TAC ACT GAC AAA CAC GGA GAA GTCTGC CCT GCT GGC 774 Gln Ala Phe Gln Tyr Thr Asp Lys His Gly Glu Val CysPro Ala Gly 235 240 245 TGG AAA CCT GGT AGT GAA ACA ATA ATC CCA GAT CCAGCT GGA AAG CTG 822 Trp Lys Pro Gly Ser Glu Thr Ile Ile Pro Asp Pro AlaGly Lys Leu 250 255 260 AAG TAT TTC GAT AAA CTG AAT TGAGAAATACTTCTTCAAGT TATGATGCTT 873 Lys Tyr Phe Asp Lys Leu Asn 265 270 GAAAGTTCTCAATAAAGTTC ACGGTTTCAT TACCACAAAA AAAAA 918 271 amino acids amino acidlinear protein 2 Met Glu Ala Leu Pro Leu Leu Ala Ala Thr Thr Pro Asp HisGly Arg 1 5 10 15 His Arg Arg Leu Leu Leu Leu Pro Leu Leu Leu Phe LeuLeu Pro Ala 20 25 30 Gly Ala Val Gln Gly Trp Glu Thr Glu Glu Arg Pro ArgThr Arg Glu 35 40 45 Glu Glu Cys His Phe Tyr Ala Gly Gly Gln Val Tyr ProGly Glu Ala 50 55 60 Ser Arg Val Ser Val Ala Asp His Ser Leu His Leu SerLys Ala Lys 65 70 75 80 Ile Ser Lys Pro Ala Pro Tyr Trp Glu Gly Thr AlaVal Ile Asp Gly 85 90 95 Glu Phe Lys Glu Leu Lys Leu Thr Asp Tyr Arg GlyLys Tyr Leu Val 100 105 110 Phe Phe Phe Tyr Pro Leu Asp Phe Thr Phe ValCys Pro Thr Glu Ile 115 120 125 Ile Ala Phe Gly Asp Arg Leu Glu Glu PheArg Ser Ile Asn Thr Glu 130 135 140 Val Val Ala Cys Ser Val Asp Ser GlnPhe Thr His Leu Ala Trp Ile 145 150 155 160 Asn Thr Pro Arg Arg Gln GlyGly Leu Gly Pro Ile Arg Ile Pro Leu 165 170 175 Leu Ser Asp Leu Thr HisGln Ile Ser Lys Asp Tyr Gly Val Tyr Leu 180 185 190 Glu Asp Ser Gly HisThr Leu Arg Gly Leu Phe Ile Ile Asp Asp Lys 195 200 205 Gly Ile Leu ArgGln Ile Thr Leu Asn Asp Leu Pro Val Gly Arg Ser 210 215 220 Val Asp GluThr Leu Arg Leu Val Gln Ala Phe Gln Tyr Thr Asp Lys 225 230 235 240 HisGly Glu Val Cys Pro Ala Gly Trp Lys Pro Gly Ser Glu Thr Ile 245 250 255Ile Pro Asp Pro Ala Gly Lys Leu Lys Tyr Phe Asp Lys Leu Asn 260 265 27029 base pairs nucleic acid single linear other nucleic acid /desc =“PRIMER” 3 GCGCGGATCC ATGGAGGCGC TGCCCTGCT 29 23 base pairs nucleic acidsingle linear other nucleic acid /desc = “PRIMER” 4 CGCCCATGGAGGCGCTGCCC CTG 23 25 base pairs nucleic acid single linear other nucleicacid /desc = “PRIMER” 5 CGCCCATGGC TGGAGCTGTG CAGGG 25 38 base pairsnucleic acid single linear other nucleic acid /desc = “PRIMER” 6CGCGTCTAGA TCAATTCAGT TTATCGAAAT ACTTCAGC 38 26 base pairs nucleic acidsingle linear other nucleic acid /desc = “PRIMER” 7 GCGCGGATCCGCTGGAGCTG TGCAGG 26 26 base pairs nucleic acid single linear othernucleic acid /desc = “PRIMER” 8 CGCGGATCCC GAGGCGCTGC CCCTGC 26 31 basepairs nucleic acid single linear other nucleic acid /desc = “PRIMER” 9CGCGGATCCT CAATTCAGTT TATCGAAATA C 31 33 base pairs nucleic acid singlelinear other nucleic acid /desc = “PRIMER” 10 CGCGGATCCG CCATCATGGAGGCGCTGCCC CTG 33 31 base pairs nucleic acid single linear other nucleicacid /desc = “PRIMER” 11 CGCGGATCCT CAATTCAGTT TATCGAAATC A 31 25 basepairs nucleic acid single linear other nucleic acid /desc = “PRIMER” 12GCGCGGATCC ACCATGGAGG CGCTG 25 52 base pairs nucleic acid single linearother nucleic acid /desc = “PRIMER” 13 GCGCTCTAGA TCAAGCGTAG TCTGGGACGTCGTATGGGTA ATTCAGTTTA TC 52 199 amino acids amino acid <Unknown> linearprotein 14 Met Ser Ser Gly Asn Ala Lys Ile Gly His Pro Ala Pro Asn PheLy 1 5 10 15 Ala Thr Ala Val Met Pro Asp Gly Gln Phe Lys Asp Ile Ser LeuSe 20 25 30 Asp Tyr Lys Gly Lys Tyr Val Val Phe Phe Phe Tyr Pro Leu AspPh 35 40 45 Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg AlaGl 50 55 60 Glu Phe Lys Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val AspSe 65 70 75 80 His Phe Cys His Leu Ala Trp Val Asn Thr Pro Lys Lys GlnGly Gl 85 90 95 Leu Gly Pro Met Asn Ile Pro Leu Val Ser Asp Pro Lys ArgThr Il 100 105 110 Ala Gln Asp Tyr Gly Val Leu Lys Ala Asp Glu Gly IleSer Phe Ar 115 120 125 Gly Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu ArgGln Ile Thr Va 130 135 140 Asn Asp Pro Pro Cys Cys Arg Ser Val Asp GluThr Leu Arg Leu Va 145 150 155 160 Gln Ala Phe Gln Phe Thr Asp Lys HisGly Glu Val Cys Pro Ala Gl 165 170 175 Trp Lys Pro Gly Ser Asp Thr IleLys Pro Asp Val Pro Lys Thr Ly 180 185 190 Glu Tyr Phe Ser Lys Gln Lys195 198 amino acids amino acid <Unknown> linear protein 15 Met Ala SerGly Asn Ala Arg Ile Gly Lys Pro Ala Pro Asp Phe Ly 1 5 10 15 Ala Thr AlaVal Val Asp Gly Ala Phe Lys Glu Val Lys Leu Ser As 20 25 30 Tyr Lys GlyLys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp Phe Th 35 40 45 Phe Val CysPro Thr Glu Ile Ile Ala Phe Ser Asn Arg Ala Glu As 50 55 60 Phe Arg LysLeu Gly Cys Glu Val Leu Gly Val Ser Val Asp Ser Gl 65 70 75 80 Phe AsnHis Leu Ala Trp Ile Asn Thr Pro Arg Lys Glu Gly Gly Le 85 90 95 Gly ProLeu Asn Ile Pro Leu Leu Gly Asp Val Thr Arg Arg Leu Se 100 105 110 GluAsp Tyr Gly Val Leu Lys Thr Asp Glu Gly Ile Ala Tyr Arg Gl 115 120 125Leu Phe Ile Ile Asp Gly Lys Gly Val Leu Arg Gln Ile Thr Val As 130 135140 Asp Leu Pro Val Gly Arg Ser Val Asp Glu Ala Leu Arg Leu Val Gl 145150 155 160 Ala Phe Gln Tyr Thr Asp Glu His Gly Glu Val Cys Pro Ala GlyTr 165 170 175 Lys Pro Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Asp SerLys Gl 180 185 190 Tyr Phe Ser Lys His Asn 195 257 amino acids aminoacid <Unknown> linear protein 16 Met Ala Ala Ala Ala Gly Arg Leu Leu TrpSer Ser Val Ala Arg Gl 1 5 10 15 Ala Ser Ala Ile Ser Arg Ser Ile Ser AlaSer Thr Val Leu Arg Pr 20 25 30 Val Ala Ser Arg Arg Thr Cys Leu Thr AspIle Leu Trp Ser Ala Se 35 40 45 Ala Gln Gly Lys Ser Ala Phe Ser Thr SerSer Ser Phe His Thr Pr 50 55 60 Ala Val Thr Gln His Ala Pro Tyr Phe LysGly Thr Ala Val Val As 65 70 75 80 Gly Glu Phe Lys Glu Leu Ser Leu AspAsp Phe Lys Gly Lys Tyr Le 85 90 95 Val Leu Phe Phe Tyr Pro Leu Asp PheThr Phe Val Cys Pro Thr Gl 100 105 110 Ile Val Ala Phe Ser Asp Lys AlaAsn Glu Phe His Asp Val Asn Cy 115 120 125 Glu Val Val Ala Val Ser ValAsp Ser His Phe Ser His Leu Ala Tr 130 135 140 Ile Asn Thr Pro Arg LysAsn Gly Gly Leu Gly His Met Asn Ile Th 145 150 155 160 Leu Leu Ser AspIle Thr Lys Gln Ile Ser Arg Asp Tyr Gly Val Le 165 170 175 Leu Glu SerAla Gly Ile Ala Leu Arg Gly Leu Phe Ile Ile Asp Pr 180 185 190 Asn GlyVal Val Lys His Leu Ser Val Asn Asp Leu Pro Val Gly Ar 195 200 205 SerVal Glu Glu Thr Leu Arg Leu Val Lys Ala Phe Gln Phe Val Gl 210 215 220Thr His Gly Glu Val Cys Pro Ala Asn Trp Thr Pro Glu Ser Pro Th 225 230235 240 Ile Lys Pro Ser Pro Thr Ala Ser Lys Glu Tyr Phe Glu Lys Val Hi245 250 255 Gln 199 amino acids amino acid <Unknown> linear protein 17Met Ser Ser Gly Asn Ala Lys Ile Gly Tyr Pro Ala Pro Asn Phe Ly 1 5 10 15Ala Thr Ala Val Met Pro Asp Gly Gln Phe Lys Asp Ile Ser Leu Se 20 25 30Glu Tyr Lys Gly Lys Tyr Val Val Phe Phe Phe Tyr Pro Leu Asp Ph 35 40 45Thr Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp Arg Ala As 50 55 60Glu Phe Lys Lys Leu Asn Cys Gln Val Ile Gly Ala Ser Val Asp Se 65 70 7580 His Phe Cys His Leu Ala Trp Ile Asn Thr Pro Lys Lys Gln Gly Gl 85 9095 Leu Gly Pro Met Asn Ile Pro Leu Ile Ser Asp Pro Lys Arg Thr Il 100105 110 Ala Gln Asp Tyr Gly Val Leu Lys Ala Asp Glu Gly Ile Ser Phe Ar115 120 125 Gly Leu Phe Ile Ile Asp Asp Lys Gly Ile Leu Arg Gln Ile ThrIl 130 135 140 Asn Asp Leu Pro Val Gly Arg Ser Val Asp Glu Ile Ile ArgLeu Va 145 150 155 160 Gln Ala Phe Gln Phe Thr Asp Lys His Gly Glu ValCys Pro Ala Gl 165 170 175 Trp Lys Pro Gly Ser Asp Thr Ile Lys Pro AspVal Asn Lys Ser Ly 180 185 190 Glu Tyr Phe Ser Lys Gln Lys 195

What is claimed is:
 1. An isolated antibody or portion thereof thatspecifically binds to a protein whose sequence consists of amino acidresidues +31 to +271 of SEQ ID NO:2.
 2. The antibody or portion thereofof claim 1 wherein said protein specifically bound by said antibody orportion thereof is glycosylated.
 3. The antibody or portion thereof ofclaim 1 which is a monoclonal antibody.
 4. The antibody or portionthereof of claim 1 which is a polyclonal antibody.
 5. The antibody orportion thereof of claim 1 which is a chimeric antibody.
 6. The antibodyor portion thereof of claim 1 which is a single chain antibody.
 7. Theantibody or portion thereof of claim 1 which is a Fab fragment.
 8. Theantibody or portion thereof of claim 1 which is labeled.
 9. The antibodyof claim 8 wherein the label is selected from the group consisting of:(a) an enzyme label; (b) a radioisotope; and (c) a fluorescent label.10. A composition comprising the antibody or portion thereof of claim 1and a carrier.
 11. The composition of claim 10, wherein the antibody orportion thereof is a monoclonal antibody.
 12. The composition of claim10, wherein the antibody or portion thereof is a polyclonal antibody.13. The composition of claim 10, wherein the antibody or portion thereofis a chimeric antibody.
 14. The composition of claim 10, wherein theantibody or portion thereof is a single chain antibody.
 15. Thecomposition of claim 10, wherein the antibody or portion thereof is aFab fragment.
 16. The composition of claim 10, wherein the antibody orportion thereof is labeled.
 17. The composition of claim 16 wherein thelabel is selected from the group consisting of: (a) an enzyme label; (b)a radioisotope; and (c) a fluorescent label.
 18. An isolated cell thatproduces the antibody or portion thereof of claim
 1. 19. A hybridomathat produces the antibody of claim
 1. 20. A hybridoma that produces theantibody of claim
 3. 21. A method of detecting NKEF C protein in abiological sample comprising: (a) contacting the biological sample withthe antibody or portion thereof of claim 1; and (b) detecting the NKEF Cprotein in the biological sample.
 22. The method of claim 21 wherein theantibody is a monoclonal antibody.
 23. The method of claim 21 whereinthe antibody is a polyclonal antibody.
 24. The method of claim 21wherein the antibody is a chimeric antibody.
 25. The method of claim 21wherein the antibody is a single chain antibody.
 26. The method of claim21 wherein the antibody is a Fab fragment.
 27. The method of claim 21wherein the antibody is a labeled antibody.
 28. The method of claim 27wherein the label is selected from the group consisting of: (a) anenzyme label; (b) a radioisotope; and (c) a fluorescent label.
 29. Anisolated antibody or portion thereof produced by immunizing an animalwith a protein whose sequence comprises amino acid residues +31 to +271of SEQ ID NO:2; wherein said antibody or portion thereof specificallybinds to the amino acid sequence of SEQ ID NO:2.
 30. An isolatedantibody or portion thereof that specifically binds to a proteinselected from the group consisting of: (a) a protein whose sequenceconsists of amino acid residues +1 to +271 of SEQ ID NO:2; (b) a proteinwhose sequence consists of at least 30 contiguous amino acid residues ofSEQ ID NO:2; and (c) a protein whose sequence consists of at least 50contiguous amino acid residues of SEQ ID NO:2.
 31. The isolated antibodyor portion thereof of claim 30, that specifically binds protein (a). 32.The isolated antibody or portion thereof of claim 30, that specificallybinds protein (b).
 33. The isolated antibody or portion thereof of claim30, that specifically binds protein (c).
 34. The isolated antibody orportion thereof of claim 30, wherein said protein specifically bound bysaid isolated antibody or portion thereof is glycosylated.
 35. Theisolated antibody or portion thereof of claim 30 which is a monoclonalantibody.
 36. The isolated antibody or portion thereof of claim 30 whichis a polyclonal antibody.
 37. The isolated antibody or portion thereofof claim 30, which is a chimeric antibody.
 38. The isolated antibody orportion thereof of claim 30 which is a single chain antibody.
 39. Theisolated antibody or portion thereof of claim 30 which is a Fabfragment.
 40. The antibody or portion thereof of claim 30 which islabeled.
 41. The antibody of claim 40 wherein the label is selected fromthe group consisting of: (a) an enzyme label; (b) a radioisotope; and(c) a fluorescent label.
 42. A composition comprising the isolatedantibody or portion thereof of claim 30 and a carrier.
 43. Thecomposition of claim 42, wherein the isolated antibody or portionthereof is a monoclonal antibody.
 44. The composition of claim 42,wherein the isolated antibody or portion thereof is a polyclonalantibody.
 45. The composition of claim 42, wherein the isolated antibodyor portion thereof is a chimeric antibody.
 46. The composition of claim42, wherein the isolated antibody or portion thereof is a single chainantibody.
 47. The composition of claim 42, wherein the isolated antibodyor portion thereof is a Fab fragment.
 48. The composition of claim 42,wherein the antibody or portion thereof is labeled.
 49. The compositionof claim 48 wherein the label is selected from the group consisting of:(a) an enzyme label; (b) a radioisotope; and (c) a fluorescent label.50. An isolated cell that produces the antibody of claim
 30. 51. Ahybridoma that produces the antibody of claim
 30. 52. A hybridoma thatproduces the antibody of claim
 35. 53. A method of assaying NKEF Cprotein in a biological sample comprising: (a) contacting the biologicalsample with the isolated antibody or portion thereof of claim 30; and(b) detecting NKEF C protein in the biological sample.
 54. The method ofclaim 53 wherein the isolated antibody or portion thereof is amonoclonal antibody.
 55. The method of claim 53 wherein the isolatedantibody or portion thereof is a polyclonal antibody.
 56. The method ofclaim 53 wherein the isolated antibody or portion thereof is a chimericantibody.
 57. The method of claim 53 wherein the isolated antibody orportion thereof is a single chain antibody.
 58. The method of claim 53wherein the antibody is a Fab fragment.
 59. The method of claim 53wherein the antibody is a labeled antibody.
 60. The method of claim 59wherein the label is selected from the group consisting of: (a) anenzyme label; (b) a radioisotope; and (c) a fluorescent label.
 61. Anantibody or portion thereof produced by immunizing an animal with aprotein selected from the group consisting of: (a) a protein whosesequence comprises amino acid residues +1 to +271 of SEQ ID NO:2; (b) aprotein whose sequence comprises 30 contiguous amino acid residues ofSEQ ID NO:2; and (c) a protein whose sequence comprises 50 contiguousamino acid residues of SEQ ID NO:2; wherein said antibody or portionthereof specifically binds to the amino acid sequence of SEQ ID NO:2.62. The antibody or portion thereof of claim 61 produced by immunizingan animal with protein (a).
 63. The antibody or portion thereof of claim61 produced by immunizing an animal with protein (b).
 64. The antibodyor portion thereof of claim 61 produced by immunizing an animal withprotein (c).
 65. An isolated antibody or portion thereof thatspecifically binds to a protein whose sequence consists of the aminoacid sequence of the mature form of the polypeptide encoded by the cDNAcontained in ATCC® Deposit No.
 97157. 66. The antibody or portionthereof of claim 65 wherein said protein specifically bound by saidantibody or portion thereof is glycosylated.
 67. The antibody or portionthereof of claim 65 which is a monoclonal antibody.
 68. The antibody orportion thereof of claim 65 which is a polyclonal antibody.
 69. Theantibody or portion thereof of claim 65 which is a chimeric antibody.70. The antibody or portion thereof of claim 65 which is a single chainantibody.
 71. The antibody or portion thereof of claim 65 which is a Fabfragment.
 72. The antibody or portion thereof of claim 65 which islabeled.
 73. The antibody of claim 72 wherein the label is selected fromthe group consisting of: (a) an enzyme label; (b) a radioisotope; and(c) a fluorescent label.
 74. A composition comprising the antibody orportion thereof of claim 65 and a carrier.
 75. The composition of claim74, wherein the antibody or portion thereof is a monoclonal antibody.76. The composition of claim 74, wherein the antibody or portion thereofis a chimeric antibody.
 77. The composition of claim 74, wherein theantibody or portion thereof is a single chain antibody.
 78. Thecomposition of claim 74, wherein the antibody or portion thereof is aFab fragment.
 79. The composition of claim 74, wherein the antibody orportion thereof is labeled.
 80. The composition of claim 79 wherein thelabel is selected from the group consisting of: (a) an enzyme label; (b)a radioisotope; and (c) a fluorescent label.
 81. An isolated cell thatproduces the antibody of claim
 65. 82. A hybridoma that produces theantibody of claim
 65. 83. A hybridoma that produces the antibody ofclaim
 67. 84. A method of detecting NKEF C protein in a biologicalsample comprising: (a) contacting the biological sample with theantibody or portion thereof of claim 65; and (b) detecting the NKEF Cprotein in the biological sample.
 85. The method of claim 84 wherein theantibody is a monoclonal antibody.
 86. The method of claim 84 whereinthe antibody is a polyclonal antibody.
 87. The method of claim 84wherein the antibody is a chimeric antibody.
 88. The method of claim 84wherein the antibody is a single chain antibody.
 89. The method of claim84 wherein the antibody is a Fab fragment.
 90. The method of claim 84wherein the antibody is a labeled antibody.
 91. The method of claim 90wherein the label is selected from the group consisting of: (a) anenzyme label; (b) a radioisotope; and (c) a fluorescent label.
 92. Anisolated antibody or portion thereof produced by immunizing an animalwith a protein whose sequence comprises the amino acid sequence of themature form of the polypeptide encoded by the cDNA contained in ATCC®Deposit No. 97157; wherein said antibody or portion thereof specificallybinds to the amino acid sequence of the polypeptide encoded by the cDNAcontained in ATCC® Deposit No.
 97103. 93. An isolated antibody orportion thereof that specifically binds to a protein selected from thegroup consisting of: (a) a protein whose sequence consists of the aminoacid sequence of the polypeptide encoded by the cDNA contained in ATCC®Deposit No. 97157; (b) a protein whose sequence consists of 30contiguous amino acid residues of a polypeptide encoded by the cDNAcontained in ATCC® Deposit No. 97157; and (c) a protein whose sequenceconsists of 50 contiguous amino acid residues of a polypeptide encodedby the cDNA contained in ATCC® Deposit No.
 97157. 94. The isolatedantibody or portion thereof of claim 93 that specifically binds protein(a).
 95. The isolated antibody or portion thereof of claim 93 thatspecifically binds protein (b).
 96. The isolated antibody or portionthereof of claim 93 that specifically binds protein (c).
 97. Theisolated antibody or portion thereof of claim 93, wherein said proteinspecifically bound by said antibody or portion thereof is glycosylated.98. The isolated antibody or portion thereof of claim 93, which is amonoclonal antibody.
 99. The isolated antibody or portion thereof ofclaim 93, which is a polyclonal antibody.
 100. The isolated antibody orportion thereof of claim 93, which is a chimeric antibody.
 101. Theisolated antibody or portion thereof of claim 93 which is a single chainantibody.
 102. The isolated antibody or portion thereof of claim 93which is a Fab fragment.
 103. The isolated antibody or portion thereofof claim 93 which is labeled.
 104. The isolated antibody or portionthereof of claim 103 wherein the label is selected from the groupconsisting of: (a) an enzyme label; (b) a radioisotope; and (c) afluorescent label.
 105. A composition comprising the isolated antibodyor portion thereof of claim 93 and a carrier.
 106. The composition ofclaim 105, wherein the antibody or portion thereof is a monoclonalantibody.
 107. The composition of claim 105, wherein the antibody orportion thereof is a polyclonal antibody.
 108. The composition of claim105, wherein the antibody or portion thereof is a chimeric antibody.109. The composition of claim 105, wherein the antibody or portionthereof is a single chain antibody.
 110. The composition of claim 105,wherein the antibody or portion thereof is a Fab fragment.
 111. Thecomposition of claim 105, wherein the antibody or portion thereof islabeled.
 112. The composition of claim 111, wherein the label isselected from the group consisting of: (a) an enzyme label; (b) aradioisotope; and (c) a fluorescent label.
 113. An isolated cell thatproduces the isolated antibody or portion thereof of claim
 93. 114. Ahybridoma that produces the antibody of claim
 93. 115. A hybridoma thatproduces the antibody of claim
 98. 116. A method of assaying NKEF Cprotein in a biological sample comprising: (a) contacting the biologicalsample from a test subject with the isolated antibody or portion thereofof claim 93; and (b) detecting NKEF C protein in the biological sample.117. The method of claim 116, wherein the antibody or portion thereof isa monoclonal antibody.
 118. The method of claim 116, wherein theantibody or portion thereof is a polyclonal antibody.
 119. The method ofclaim 116, wherein the antibody or portion thereof is a chimericantibody.
 120. The method of claim 116, wherein the antibody or portionthereof is a single chain antibody.
 121. The method of claim 116,wherein the antibody or portion thereof is a Fab fragment.
 122. Themethod of claim 116, wherein the antibody or portion thereof is labeled.123. The method of claim 122, wherein the label is selected from thegroup consisting of: (a) an enzyme label; (b) a radioisotope; and (c) afluorescent label.
 124. An antibody or portion thereof produced byimmunizing an animal with a protein selected from the group consistingof: (a) a protein whose sequence comprises the amino acid sequence ofthe polypeptide encoded by the cDNA contained in ATCC® Deposit No.97157; (b) a protein whose sequence comprises at least 30 contiguousamino acid residues of a polypeptide encoded by the cDNA contained inATCC® Deposit No. 97157; and (c) a protein whose sequence comprises atleast 50 contiguous amino acid residues of a polypeptide encoded by thecDNA contained in ATCC® Deposit No. 97157; wherein said antibody orportion thereof specifically binds to the polypeptide encoded by thecDNA contained in ATCC® Deposit No.
 97157. 125. (New) The antibody orportion thereof of claim 124 produced by immunizing an animal withprotein (a).
 126. (New) The antibody or portion thereof of claim 124produced by immunizing an animal with protein (b).
 127. (New) Theantibody or portion thereof of claim 124 produced by immunizing ananimal with protein (c).
 128. A method of treating a patient having needof a reduced level of NKEF C protein, comprising administering to saidpatient the antibody or portion thereof of claim
 1. 129. The method ofclaim 128, wherein the antibody is a monoclonal antibody.
 130. A methodof treating a patient having need of a reduced level of NKEF C protein,comprising administering to said patient the antibody or portion thereofof claim
 30. 131. A method of treating a patient having need of areduced level of NKEF C protein, comprising administering to saidpatient the antibody or portion thereof of claim
 65. 132. A method oftreating a patient having need of a reduced level of NKEF C protein,comprising administering to said patient the antibody or portion thereofof claim 93.